CRISPR/Cas9-assisted gene editing reveals that EgPKS, a polyketide synthase, is required for the biosynthesis of preussomerins in Edenia gomezpompae SV2.

Edenia gomezpompae, an endophytic fungus derived from plants, produced a diverse array of preussomerins, a type of spirobisnaphthalenes featuring two spiroketal groups, which exhibited significant antibacterial, antifungal, and cytotoxic activities. Structurally, the biosynthesis of preussomerins might be related to the biosynthesis of 1,8-dihydroxynaphthalene (DHN), a precursor of DHN-melanin. However, the absence of efficient gene-editing tools for E. gomezpompae has hindered the biosynthetic study of preussomerins. In this study, we developed a CRISPR/Cas9-based gene editing system for E. gomezpompae SV2 that was isolated from the stem of Setaria viridis, by utilizing the endogenous U6 snRNA promoter to drive sgRNA expression. Using this system, we successfully disrupted the polyketide synthase (PKS)-encoding gene, Egpks, a putative 1,3,6,8-tetrahydroxynaphthalene synthase gene involved in the biosynthesis of DHN-melanin, with an editing efficiency up to 92% and a knockout efficiency of 71% when employing the U6 snRNA-3 promoter. Furthermore, the disrupted mutant (∆Egpks) displayed white hyphae and lost the ability to produce preussomerins. These results provided a foundational tool for genetic manipulation in E. gomezpompae and revealed the role of EgPKS in the biosynthesis of preussomerin-type spirobisnaphthalenes.